The major goal of this project is to determine the basic functional mechanism of cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain. CcO is membrane-bound and has the dual function of reducing O2 to H2O to maintain electron flow to enable oxidative phosphorylation, and of coupling the oxygen reduction chemistry to proton translocation across the inner mitochondrial membrane, to generate a proton gradient. The enzyme plays an indispensable role in mammalian physiology because essentially all vital organs depend on aerobic metabolism. Despite years of intensive studies on CcO, the understanding of its catalytic processes is still incomplete and its mechanism of proton translocation remains unclear. We propose a variety of experiments to delineate the oxygen reduction chemistry and aim to shed new light on the coupled proton translocation. In order to identify the catalytic intermediates, new rapid mixing, laser photolysis and freeze trapping techniques will be applied to studies of both the mammalian and the bacterial forms of the enzyme. Full characterization will be done with resonance Raman scattering, optical absorption and electron paramagnetic resonance spectroscopies. One of the major focuses will be to determine the properties of the still controversial short-lived peroxo and ferryl intermediates. Resonance Raman and optical absorption spectroscopy will also be used in conjunction with a home-built continuous flow apparatus to monitor the time-dependent population of the reactive intermediates present during single as well as multiple turnover of the enzyme. To determine the role of specific residues in the functional processes of the enzyme, site directed mutants of CcO from bacteria will be expressed and analyzed by this multifaceted approach. In addition, the oxygen chemistry in a new class of model complexes, a mutant myoglobin with a binuclear center engineered in it to mimic the catalytic site of CcO, will be studied to elucidate the role of the binuclear heme-copper center. The energy transduction and proton translocation mechanism of CcO will be constructed, tested and refined.